The Asian citrus psyllid (Diaphorina citri) is the insect vector responsible for the worldwide spread of ‘Candidatus Liberibacter asiaticus’ (CLas), the bacterial pathogen associated with citrus greening disease. Developmental changes in the insect vector impact pathogen transmission, such that D. citri transmission of CLas is more efficient when bacteria are acquired by nymphs when compared with adults. We hypothesize that expression changes in the D. citri immune system and commensal microbiota occur during development and regulate vector competency. In support of this hypothesis, more proteins, with greater fold changes, were differentially expressed in response to CLas in adults when compared with nymphs, including insect proteins involved in bacterial adhesion and immunity. Compared with nymphs, adult insects had a higher titre of CLas and the bacterial endosymbionts Wolbachia, Profftella and Carsonella. All Wolbachia and Profftella proteins differentially expressed between nymphs and adults are upregulated in adults, while most differentially expressed Carsonella proteins are upregulated in nymphs. Discovery of protein interaction networks has broad applicability to the study of host–microbe relationships. Using protein interaction reporter technology, a D. citri haemocyanin protein highly upregulated in response to CLas was found to physically interact with the CLas coenzyme A (CoA) biosynthesis enzyme phosphopantothenoylcysteine synthetase/decarboxylase. CLas pantothenate kinase, which catalyses the rate-limiting step of CoA biosynthesis, was found to interact with a D. citri myosin protein. Two Carsonella enzymes involved in histidine and tryptophan biosynthesis were found to physically interact with D. citri proteins. These co-evolved protein interaction networks at the host–microbe interface are highly specific targets for controlling the insect vector responsible for the spread of citrus greening.

Genetic variability in insect vectors is valuable to study vector competence determinants and to select non-vector populations that may help reduce the spread of vector-borne pathogens. We collected and tested vector competency of 15 isofemale lines of Asian citrus psyllid, Diaphorina citri, vector of ‘Candidatus Liberibacter asiaticus’ (CLas). CLas is associated with huanglongbing (citrus greening), the most serious citrus disease worldwide. D. citri adults were collected from orange jasmine (Murraya paniculata) hedges in Florida, and individual pairs (females and males) were caged on healthy Murraya plants for egg laying. The progeny from each pair that tested CLas-negative by qPCR were maintained on Murraya plants and considered an isofemale line. Six acquisition tests on D. citri adults that were reared as nymphs on CLas-infected citrus, from various generations of each line, were conducted to assess their acquisition rates (percentage of qPCR-positive adults). Three lines with mean acquisition rates of 28 to 32%, were classified as ‘good’ acquirers and three other lines were classified as ‘poor’ acquirers, with only 5 to 8% acquisition rates. All lines were further tested for their ability to inoculate CLas by confining CLas-exposed psyllids for one week onto healthy citrus leaves (6–10 adults/leaf/week), and testing the leaves for CLas by qPCR. Mean inoculation rates were 19 to 28% for the three good acquirer lines and 0 to 3% for the three poor acquirer lines. Statistical analyses indicated positive correlations between CLas acquisition and inoculation rates, as well as between CLas titer in the psyllids and CLas acquisition or inoculation rates. Phenotypic and molecular characterization of one of the good and one of the poor acquirer lines revealed differences between them in color morphs and hemocyanin expression, but not the composition of bacterial endosymbionts. Understanding the genetic architecture of CLas transmission will enable the development of new tools for combating this devastating citrus disease.

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Diaphorina citri is a vector of ‘ Candidatus Liberibacter asiaticus, ’ ( C Las), associated with Huanglongbing, (HLB, or citrus greening) disease in citrus. D . citri exhibits three different color morph variants, blue, gray and yellow. Blue morphs have a greater capacity for long-distance flight as compared to non-blue morphs, but little else is known about how color morphology influences vector characteristics. In this study, we show that the color morphology of the insect is derived from pigmented cells of the fat body. Blue morphs acquire a lower level of C Las in their bodies from infected trees as compared to their gray and yellow conspecifics, referred to in this paper collectively as non-blue morphs. Accordingly, C Las titer in citrus leaves inoculated by non-blue insects was 6-fold higher than in leaves inoculated by blue insects. Blue color morphs harbored lower titers of Wolbachia and ‘ Candidatus Profftella armatura,’ two of the D . citri bacterial endosymbionts. Expression of hemocyanin, a copper-binding oxygen transport protein responsible for the blue coloration of hemolymph of other arthropods and mollusks, was previously correlated with blue color morphology and is highly up-regulated in insects continuously reared on C Las infected citrus trees. Based on our results, we hypothesized that a reduction of hemocyanin expression would reduce the D . citri immune response and an increase in the titer of C Las would be observed. Surprisingly, a specific 3-fold reduction of hemocyanin-1 transcript levels using RNA silencing in blue adult D . citri morphs had an approximately 2-fold reduction on the titer of C Las. These results suggest that hemocyanin signaling from the fat body may have multiple functions in the regulation of bacterial titers in D . citri , and that hemocyanin is one of multiple psyllid genes involved in regulating C Las titer.

An in vivo trial was conducted to evaluate the effects of Bacillus coagulans, and inulin, either separately or in combination, on lipid profile using a rat model. Thirty-two male Wistar rats were randomly divided into four groups (n=8) and fed as follows: standard diet (control), standard diet with 5% w/w long chain inulin (prebiotic), standard diet with 10 9 spores/day spores of B. coagulans by orogastric gavage (probiotic), and standard diet with 5% w/w long chain inulin and 10 9 spores/day of B. coagulans (synbiotic). Rats were fed for 30 days. Serum samples were collected 10, 20 and 30 days following onset of treatment. Total, HDL and LDL cholesterol and triglycerides concentrations were analyzed. Results of this study showed that inulin potentially affected the lipid profile. An obvious decrease in serum total cholesterol and LDL-cholesterol of rats fed with inulin in symbiotic and prebiotic groups was seen in all sampling days. Inulin fed rats also demonstrated higher levels of HDL-cholesterol concentration; however this value in probiotic and control fed rats remains without significant change. According to the results of this study, B. coagulans did not contribute to any lipid profile changes after 30 days. Thus, further in vitro investigations on the characteristic of these bacteria could be useful to gain insights into understanding the treatment of probiotics in order to achieve the maximum beneficial effect.

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Diaphorina citri is a vector of “Candidatus Liberibacter asiaticus” (CLas), associated with citrus greening disease. D. citri exhibit at least two color morphotypes, blue and non-blue, the latter including gray and yellow morphs. Blue morphs have a greater capacity for long-distance flight and transmit CLas less efficiently as compared to non-blue morphs. Differences in physiology and immunity between color morphs of the insect vector may influence disease epidemiology and biological control strategies. We evaluated the effect of CLas infection on color morph and sex-specific proteomic profiles of D. citri. Immunity-associated proteins were more abundant in blue morphs as compared to non-blue morphs but were upregulated at a higher magnitude in response to CLas infection in non-blue insects. To test for differences in color morph immunity, we measured two phenotypes: (1) survival of D. citri when challenged with the entomopathogenic fungus Beauveria bassiana and (2) microbial load of the surface and internal microbial communities. Non-blue color morphs showed higher mortality at four doses of B. bassinana, but no differences in microbial load were observed. Thus, color morph polyphenism is associated with two distinct proteomic immunity phenotypes in D. citri that may impact transmission of CLas and resistance to B. bassiana under some conditions.

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